Cisplatin-induced Hela Cell Apoptosis After The Ultrweak Chemiluminescence And Changes In The Level Of Oxygen Free Radicals

Cervical cancer is a popular malignant tumor, which brings severe threaten to female healthy and life. Chemotherapy is one of main measures to treat patients with cervical cancer. Cisplatin is a kind of drug to treat cervix carcinoma, which is used widely and efficiently. Moreover, it has both chemical drug and radiation promoting agent effects. However,clinical data suggests that the efficiency of chemotherapy for cervical cancer is very limited. The tumor cells with high drug-resistance are the major cause for tumor relapse and metastasis post-chemotherapy. Therefore, the evaluation of drug-resistance is a very important step in clinical medicine.Super ultraweak bioluminescence exist in intracellular and extracellular information dissemination, it originate in the process of biological growth and metabolism, and is an spontaneous chemiluminescence. Different cells, or different physiological and pathological states in the same cell, have different intensity of light. Some study suggest, after the treatment of drugs or drug-resistance, the intensity of light can be changed in tumor cells. Therefore, Super ultraweak bioluminescence may provide new method for the drug evaluation of sensitivity and resistance in tumor chemotherapy.In this study, we selected Hela cell of human cervix carcinoma, observed apoptosis induced by cisplatin and the change of ultro-structure intensity. Meanwhile, we also tested the level of ROS after chemical drug treatment, and elucidated the relationship between chemotherapy and super faint lighting. OBJECTIVE: To explore changes of super ultraweak bioluminescence and Oxygen Free Radicals in Hela cell following the apoptosis induced by DDP.METHODS:1.Hela cell was treated with DDP, DDP semi-inhibit concentration IC50 was evaluated by MTT method.2.HE stain, Flow Cytometry, immunohistochemistry and electric micro- scope was used to observe the change of Hela cell after the treatment of DDP.3.IFFM-D chemical lighting instrument was used to detect super faint light intensity after the treatment of DDP.4.Chemical colorimetry was used to evaluate the change of MDA, SOD and GSH after the treatment of DDP.RESULTS:1.evaluation of DDP cell toxicity: When the concentration of DDP below 3mg/L, it has no toxic effect on Hela cell(P<0.01), When the concentration of DDP above 3mg/L, its toxicity has dose-dependent effect(P<0.01).2.The change of Hela cell after drug treatment(1)After 12 h,24 h,48 h,72 h,96 h of using 3 mg /L DDP, Hela cell detached and generated apoptosis gradually.(2)Morphology of apoptosis cell seen by electric microscope was as follows: microvillus vanished in apoptosis cell, septum was found between cells, globular process were seen on membrane. Nucleolus disappeared at early stage of apoptosis. Chromatin were highly compressed, margined, deactivated and concentrated on nucleus membrane as enplaque or luniform. Nucleus membrane wrinkled, cytoplasm compressed and cracked as fragments. Organelles concentrated, endoplasmic reticulum expanded, cell membrane blebbing or pullulated, mitochondrial swelled and spine ruptured, uptake by macrophage and apoptosis body emerged.(3)Flow Cytometer: Hela cells in G2 phase increased after DDP treatment, while cells in S phase decreased(P<0.01).Apoptosis cells increased with the time change of DDP treatment: the values of 24h, 48h, 72h was(11.4±5.8, 21.8±7.9,32.5±11.6)% respectively(P<0.05).(4)Immunochemistry: P27 was not expressed on membrane of Hela in control group and expressed in experiment group, the proportion was 80%.3.after 3 mg /L DDP treatment, the intensity of Super faint lighting was decreased significantly(P<0.01).4.the activity of SOD and GSH in Hela cells were decreased after DDP treatment(P<0.05). while MDA increased(P<0.01).CONCLUSIONS:1.DDP can inhibit the growth of Hela cell. Our study showed the non-toxic dose was 3 mg /L;2.The intensity of super faint lighting decreased dramatically after DDP treatment. It may be due to the low cell proliferation speed, protein oxidation, DNA strain rupture and the decrease of intercellular metabolism;3.Free radical increased in Hela cell after DDP treatment. Overdosed free radical concentrated and injured cell function, led to cell apoptosis or death, which may be one of reasons for the decrease intensity of super faint lighting in cells.